Borrelia burgdorferi is the causative agent of human Lyme disease. The major manifestations of Lyme disease are caused by inflammatory changes due to the host immune response to the organism. The innate immune response plays a key role in mediating control of B. burgdorferi infection and in initiating inflammatory responses. Toll like receptors (TLRs) are a major family of innate pattern recognition receptors. TLR2 recognizes components of bacterial, fungal and viral products including lipoproteins, atypical lipopolysaccarides, ??glucans, hemagglutinin and glycophosphatidylinositol. It is one of the key drivers of inflammatory responses to B. burgdorferi due to recognition of its lipoproteins. Although TLR2 has long been thought to recognize ligands at the plasma surface membrane, our lab and others have shown that TLR2 can also signal from within endosomal compartments. We have recently identified a new co-receptor for TLR2, integrin ?3?1, that mediates endocytosis of both B. burgdorferi and the synthetic TLR2 ligand palmitoyl-3-Cys- Ser-(Lys)4 (Pam3CSK4). TLR2 signaling in response to B. burgdorferi or Pam3CSK4 while similar in many respects, also differs in the downstream cytokines induced. Our preliminary data suggests that B. burgdorferi and Pam3CSK4 are trafficked into different endosomal compartments during the endocytic process. Studies of other receptors have suggested that subcellular localization may affect signaling pathway activation. In this proposal, we will determine the mechanisms by which B. burgdorferi and Pam3CSK4 are recognized by TLR2 and integrin ????. In Aim 1, we will use an RNAi library to identify molecules involved in uptake and trafficking of B. burgdorferi and Pam3CSK4. We will then use confocal fluorescent microscopy on fixed and live cells to confirm the mechanisms of processing of B. burgdorferi and Pam3CSK4, and to identify signaling compartments for each type of stimulus. In Aim 2, we will use 2-dimensional difference gel electrophoresis (DIGE) to identify differences in signaling pathway activation between B. burgdorferi and Pam3CSK4 and to understand how integrin ???? mediated endocytosis affects signaling and trafficking of each stimulus. The mechanisms by which subcellular localization affects TLR signaling are just beginning to be explored. The results of these studies will be important for understanding the differences in processing of synthetic ligands from live bacteria and determining the role of subcellular localization of receptors and ligands into specific compartments on inflammatory signaling and cytokine release. PUBLIC HEALTH RELEVANCE: The innate immune system is one of the most important lines of host defense against microbial invaders, but it can also cause illness when stimulated inappropriately or too vigorously. In this proposal, we examine the mechanisms by which a specific component of the innate immune system, toll-like receptor 2, recognizes its targets and causes inflammation. A better understanding of the workings of innate immunity is important for developing therapies for diseases such as Lyme disease where either an ineffective or an over-exuberant immune response causes the symptoms of illness.